Dysfunctional TLR1 reduces the therapeutic efficacy of chemotherapy by attenuating HMGB1-mediated antitumor immunity in locally advanced colorectal cancer

Regional lymph node metastasis is an important predictor for survival outcome and an indicator for postoperative adjuvant chemotherapy in patients with colorectal cancer. Even with advances in adjuvant chemotherapeutic regimens, 5-year distant metastasis and survival rates are still unsatisfactory. Here, we evaluate the clinical significance of polymorphisms in receptors for HMGB1, which is the hallmark of chemotherapy-induced immunogenic cell death, in patients with stage II–III colon carcinoma (COAD). We found that high cytosolic HMGB1 is elicited in stage III COAD patients who received adjuvant chemotherapy. Patients with the TLR1-N248S polymorphism (rs4833095), which causes loss-of-function in HMGB1-mediated TLR1–TLR2 signaling, may influence the therapeutic efficacy of adjuvant chemotherapy, leading to a high risk of distant metastasis within 5 years [HR = 1.694, 95% CI = 1.063–2.698, p = 0.027], suggesting that TLR1-N248S is an independent prognostic factor for locally advanced colon carcinoma patients. We found that defective TLR1 impaired TLR1/2 signaling during dendritic cell (DC) maturation for the antitumor immune response under immunogenic chemotherapy oxaliplatin (OXP) treatment. Defective TLR1 on DCs impaired their maturation ability by HMGB1 and reduced the secretion of IFNγ from T cells to eradicate tumor cells in vitro. Moreover, systemic inhibition of TLR1/2 dramatically reduced the tumor-infiltrating immune cells by OXP treatment, leading to poor therapeutic response to OXP. In contrast, administration of a TLR1/2 agonist synergistically increased the benefit of OXP treatment and triggered a high density of tumor-infiltrating immune cells. We also observed that fewer tumor-infiltrating cytotoxic T lymphocytes were located within the tumor microenvironment in patients bearing the TLR1-N248S polymorphism. Overall, our results suggest that dysfunctional TLR1 may reduce the therapeutic response to adjuvant chemotherapy by impairing HMGB1-mediated DC maturation and attenuating the antitumor immune response in locally advanced colon carcinoma patients.


Tissue microarray (TMA) construction and immunohistochemistry
Stage II-III colon carcinoma patients who were diagnosed and treated between 2006 and 2014 at China Medical University Hospital were enrolled in our cohort 25,26 .The TMA included resected primary tumor tissue and their corresponding normal mucosa specimens, which was approved by the Institutional Review Board (IRB) of China Medical University Hospital [Protocol number: CMUH105-REC2-073].The collection of specimens and methods was carried out in accordance with the approval guidelines from the committee.Briefly, central areas of the resected primary tumor were marked on hematoxylin/eosin-stained slides by pathology, and the corresponding area on the matching formalin-fixed, paraffin-embedded tissue (FFPE) was then identified by punching 2-mm-diameter tissue cylinders.The criteria for TMA spots were at least 50% tumor cells 27 .
Immunohistochemistry (IHC) was performed using 3-μm-thick TMA sections with the indicated antibodies, followed by an HRP-conjugated avidin biotin complex (ABC) Kit (Vector Laboratories, CA, USA), incubation with HRP substrate DAB chromogen (Vector Laboratories) and counterstaining in hematoxylin 28,29 .The immune cell markers CD8 (ab217344, Abcam) were counted at 400× magnification under a microscope.The average number of TILs in five high-power fields (400× magnification) was scored by two pathologists.Cytosolic HMGB1 (ab18256, Abcam) expression was based on the H-score 25,30 , according to the intensity and percentage of positive cells for histoscore (H-score), which was calculated by a semiquantitative assessment of both the intensity of the staining (0: negative staining; 1: weak; 2: moderate; and 3: strong) and the percentage of immunopositive cells.The range of the H-score was from 0 to 300.The cytosolic HMGB1expression status was categorized as low or high according to the median value of the H-score.

Genomic DNA extraction and SNP genotyping
Genomic DNA from nontumor tissues of rectal cancer patients was extracted from two 5-μm-thick FFPE slides using a QIAamp® DNA FFPE Extraction Kit (QIAGEN GmbH, Hilden, Germany).For SNP genotyping, 10 ng of total genomic DNA was used for PCR amplification and was performed using the iPLEX® HS panel on the MassARRAY® System (Agena Bioscience, San Diego, CA, USA), which employs matrix-assisted laser desorption/ ionization time-of-fight mass spectrometry for amplicon detection (MALDITOF-MS; SpectroACQUIRE, Agena Bioscience).Primers designed (Supplementary Table S1) for PCR amplification of specific polymorphisms and extension reactions were prepared using MassARRAY® Assay Design Version 3.1 software (Agena Bioscience, San Diego, CA, USA).Following PCR, SAP addition, and the iPLEX HS® extension reaction, the samples were desalted by resin treatment for 15 min, spotted onto SpectroCHIP® Arrays (Agena Bioscience, San Diego, CA), analyzed by mass spectrometry, and ultimately interpreted using SpectroTYPER v4.0 software (Agena Bioscience, San Diego, CA).The characteristics of the selected polymorphisms are shown in Table S1.

THP1-derived immature DCs and colorectal cancer cell line coculture
The human monocytic leukemia cell line THP1 was cultured and maintained in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Grand Island, New York, USA), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified incubator with 95% air and 5% CO 2 .After the gene was silenced by lentivirus carrying shRNA against the indicated genes, immature DCs were generated from THP1 cells as previously described 32 .Briefly, THP1 cells were differentiated into immature DCs by adding 1500 IU/ml rhIL-4 (Sino Biological, Beijing, China) and 1500 IU/ml rhGM-CSF (Sino Biological, Beijing, China) in culture medium for at least 7 days, with cytokine-supplemented culture medium changed every 2-3 days at 37 °C in a humidified incubator with 95% air and 5% CO 2 .These THP1-iDC cells were treated with recombinant human HMGB1 (Sino Biological, Beijing, China) for 24 h.DC maturation was measured by flow cytometry.These THP1-iDC cells were also cocultured with OXP-treated HT29-GFP cells to analyze the DC maturation marker CD86 by flow cytometry.In addition, the human T-cell leukemia Jurkat cell line was coincubated with OXP-treated HT29/THP1-iDCs for 15 h to analyze the level of IFNγ.

Statistical analysis
The statistical analysis was performed by SPSS (IBM SPSS Statistics 22, WA, USA), and GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) was utilized.A two-sided p < 0.05 was used as the significance level for all tests, such as Student's t test, Pearson chi-square test and Fisher's exact test.Univariate and multivariate analyses were performed by Cox regression analysis to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs).These influential factors were adjusted in the Cox models, including sex (male vs. female), age (≥ 65 vs. < 65), pT stage (pT 3-4 vs. pT 1-2), tumor location (proximal vs. distal), tumor differentiation (poor vs. well to moderate), LVI (present vs. absent), PNI (present vs. absent) and TLR1-N248 (variant vs. WT) and TLR2 promoter (variant vs. WT).Kaplan-Meier analysis was used to assess distant metastasis-free survival (DMFS) and disease-free survival (DFS) with the survival time between surgery and events such as tumor relapse and death.

Ethical approval
This study was reviewed and approved by the Internal Review Board (IRB) of China Medical University Hospital [Protocol number: CMUH105-REC2-073].The method was carried out in accordance with the committee's approved guidelines.

Informed consent
Informed consents were obtained from all participants in the study.

Clinical characteristics and genotype of TLR1/TLR2, which are HMGB1 receptors, in stage II-III patients
Our previous studies have demonstrated that the density and cytosolic pattern of HMGB1 (cyto-HMGB1) is an independent prognostic factor in locally advanced rectal cancer patients who underwent preoperative concurrent chemoradiotherapy, suggesting that chemoradiotherapy elicits the release of HMGB1 to promote ICD and antitumor immunity 26 .In this study, we investigated whether cyto-HMGB1 is associated with survival outcomes in patients with locally advanced colon adenocarcinoma (COAD) who received adjuvant chemotherapy.As shown in Fig. 1A-C, high cyto-HMGB1 levels in tumor tissues of stage III COAD patients were associated with poor distant metastasis-free survival (DMFS) and disease-free survival (DFS).Patients with low cyto-HMGB1 had a negative correlation with higher nuclear HMGB1 expression and were associated with a more favorable survival outcome, which was consistent with previous studies 39 .These results suggest that environment-driven inflammation for cyto-HMGB1 before chemotherapy may promote tumor progression and inhibit antitumor immunity with other mechanisms, such as autophagy [39][40][41][42][43] .However, chemotherapy-induced ICD signatures may be associated with antitumor immunity and influence the risk of distant metastasis and the therapeutic efficacy of chemotherapy 8 .Our results demonstrated that cyto-HMGB1 was significantly increased in advanced-stage CRC patients after chemotherapy treatment (Fig. 1D, p = 0.0017).
In the univariate analysis of DMFS in locally advanced stage III COAD patients, the following parameters were associated with patient survival rate: age, pT stage, LVI and PNI.Moreover, patients with the TLR1-N248 polymorphism had an increased risk for lower DMFS (HR = 1.725, 95% CI = 1.084-2.745,p = 0.0215, Fig. 1E) and DFS (HR = 1.632, 95% CI = 1.121-2.395,p = 0.0296, Fig. 1F) compared with patients carrying wild-type TLR1 www.nature.com/scientificreports/(Table 3).These results indicate that the TLR1-N248 polymorphism has significant prognostic value for locally advanced stage III COAD patients.Subsequently, we examined whether the inclusion of other variables was significantly associated with the survival of locally advanced COAD.Our results showed that the TLR1-N248 polymorphism (HR = 1.694, 95% CI = 1.063-2.698,p = 0.027) was an independent prognostic factor of DMFS for locally advanced COAD patients (Table 3), indicating that the TLR1-N248 polymorphism has significant prognostic value for COAD patients with regional lymph node metastasis.These results suggested that loss-offunction in TLR1 may impair the immunogenic effect of released HMGB1 on antitumor immunity elicited by immunogenic chemotherapeutic agents such as oxaliplatin (OXP).To demonstrate that defective TLR1 impairs HMGB1-mediated TLR1/2 signaling for DC maturation by immunogenic chemotherapy, we first treated HT29 and HCT116 cells with OXP and analyzed the level of released HMGB1 (Fig. 2A).Indeed, we found that released HMGB1 was clearly detected in conditioned medium (CM) after OXP treatment (Fig. 2A).These OXP-treated CM from HT29 and HCT116 cells significantly triggered the activation of the TLR-mediated downstream, MYD88, ERK and IRF3 signaling pathway in THP1-iDC cells (Fig. 2B).However, the addition of anti-HMGB1 neutralizing antibodies clearly diminished the activation of the TLR-mediated signaling pathway, suggesting that HMGB1 released by OXP was critical to enhance DC maturation by TLR receptors.The released HMGB1 promoting DC maturation was mainly dependent on TLR2, TLR4 and RAGE for antitumor immunity 26,44,45 .Therefore, we combined several small molecule inhibitors, including FPS-ZM1 (RAGE antagonist), TLR4-IN-C34 (TLR4 antagonist) and CU-CPTT22 (TLR1/2 antagonist), with HMGB1 recombinant protein (rhHMGB1) to evaluate DC maturation.We found that blockade of TLR1/2, TLR4 and RAGE significantly decreased HMGB1-enhanced DC maturation (Fig. 2C).The level of DC maturation markers CD80 and CD86 were significantly decreased (Fig. 2C).But we found that TLR1/2 and TLR4 mainly participate in HMGB1-mediated DC maturation, compared to RAGE signaling pathway.
We then cocultured OXP-treated HT29-GFP cancer cells with THP1 shNC -iDCs and THP1 shTLR1 -iDCs for 24 h (Fig. 2G).We found that the DC maturation marker CD86 was significantly decreased in THP1 shTLR1 -iDC cells after coculture with OXP-treated HT29-GFP cells compared to THP1 shNC -iDC cells (Fig. 2G).Then, we cocultured Jurkat T lymphocytes with OXP-treated HT29/THP1-iDCs to evaluate the level of the cytotoxic marker IFNγ (Fig. 2H).As shown in Fig. 2H, we found that the level of IFNγ in Jurkat T lymphocytes was also significantly decreased in THP1 shTLR1 -iDCs (Fig. 2H).These results suggested that defective TLR1 attenuated TLR2 signaling, leading to less DC maturation and T-cell activation by immunogenic chemotherapy.

TLR1/2 signaling influences the therapeutic efficacy of chemotherapy by boosting antitumor immunity
To further validate that the therapeutic efficacy of chemotherapy was influenced by TLR1/2 signaling via antitumor immunity, we combined either the TLR1/2 antagonist CU-CPT22 or the TLR1/2 agonist Pam 3 CSK 4 with OXP (Fig. 3A).Mice were given a subcutaneous tumor challenge with CT26 for 13 days and treated with OXP with or without CU-CPT22 and Pam 3 CSK 4 .We found that tumor volume was decreased in mice receiving 5 cycles of OXP (Fig. 3B).By pharmacologic inhibition of TLR1/2 signaling, we found that the therapeutic efficacy of OXP was significantly inhibited by administration of CU-CPT22 (Fig. 3B-D).In contrast, tumor regression was markedly reduced in the OXP/Pam 3 CSK 4 group compared to mice that received OXP only (Fig. 3B-D), suggesting that activation of TLR1/2 signaling may enhance the therapeutic efficacy of OXP.
We then evaluated the immune cell profile within the tumor microenvironment (TME) by flow cytometry and IHC.The density of tumor-infiltrating CD11c + DCs and GzmB + CD8 + T cells was markedly decreased in the OXP/CU-CPT22 group (Fig. 3E-G).However, there was an increase in the OXP/Pam 3 CSK 4 group compared to the OXP group (Fig. 3E-G).By flow cytometric analysis, we found that tumor-infiltrating CD8 + T cells were also significantly increased in the OXP/Pam 3 CSK 4 group compared to the other groups (Fig. 3H,I).Blockade of TLR1/2 significantly reduced the infiltration of CD8 T cells elicited by OXP treatment (Fig. 3H,I), suggesting that activation of TLR1/2 signaling may enhance the therapeutic efficacy of OXP by boosting antitumor immunity.But we found that the density of tumor-infiltrating CD4 + T cells were significantly decreased in the OXP/ Pam 3 CSK 4 group compared to the other groups (Fig. 3I).
By evaluating the density of effector/memory CD8 cells, we found that there was a remarkable decrease in the OXP/CU-CPT22 group but an increase in the OXP/Pam 3 CSK 4 group (Fig. 4A).The tumor-infiltrating IFNγ + CD8 cells were also increased in the OXP/Pam 3 CSK 4 group (Fig. 4B,C).However, we found no significant change in immunosuppressive Foxp3 + regulatory T cells within TME (Fig. 4D).Taken together, these results showed that OXP can induce infiltration of effector/memory and cytotoxic immune cells to recognize and eradicate residual tumor cells via TLR1/2-mediated signaling, indicating that TLR1/2 is critical to enhance the therapeutic efficacy of immunogenic chemotherapy.Additionally, the density of tumor-infiltrating CD8 + T cells was significantly lower in locally advanced stage III COAD patients bearing the TLR1-N248S polymorphism (Fig. 4E).Taken together, these results showed that defective TLR1 may reduce the therapeutic efficacy of chemotherapy in locally advanced COAD patients by attenuating HMGB1-mediated antitumor immunity.

Discussion
Our data showed for the first time that TLR1-N248S was associated with the clinical outcome of patients with locally advanced stage III COAD, suggesting that this genetic defect might be associated with the risk of lymph node metastasis and poor DMFS.We found that defective TLR1 might attenuate HMGB1-mediated ICD to decrease the response to immunogenic chemotherapy, leading to a reduced antitumor response and poor therapeutic efficacy in vitro and in vivo.Furthermore, patients carrying the TLR1-N248S polymorphism had fewer The conditioned medium from OXP (25 μM)-treated HT29 and HCT116 cells was collected after 24 h of treatment.These CMs were preincubated with anti-HMGB1 antibodies (1 μg/mL) for 0.5 h and then added to THP1-iDCs, which were differentiated into immature DCs (iDCs) by IL-4 (1500 IU/ml) and GM-CSF (1500 IU/ ml) for 7 days.(C) THP1-iDCs were treated with different small molecules for 1 h, and then rhHMGB1 was added for 24 h to analyze the maturation of DCs (CD86 and CD80) by qRT-PCR (n = 3).One-way ANOVA t test, **p < 0.01.(D) TLR1 was knocked down in THP1 cells by lentivirus carrying shRNA with a spinoculation protocol.Then, THP1-iDCs were treated with rhHMGB1 for 24 h for analysis by immunoblotting.(E) THP1 shNC -iDCs and THP1 shTLR1 -iDCs were treated with rhHMGB1 for 24 h to analyze the maturation of DCs (CD86 and CD80) by qRT-PCR (n = 3).One-way ANOVA t test, **p < 0.01.(F) THP1 shNC -iDCs and THP1 shTLR1 -iDCs were treated with rhHMGB1 protein for 24 h.DC maturation CD86 was analyzed by flow cytometry (n = 3).One-way ANOVA t test, **p < 0.01.(G) The diagram scheme of coculture analysis.HT29-GFP cells were treated with OXP (20 μM) for 24 h, and then the drugs were washed to coculture with THP1 shNC -iDCs and THP1 shTLR1 -iDCs for 24 h.DC maturation of CD86 was analyzed by flow cytometry (n = 3).One-way ANOVA t test, **p < 0.01.(H) HT29 cells were treated with OXP (20 μM) for 24 h, and then the drugs were washed out and cocultured with THP1 shNC -iDCs and THP1 shTLR1 -iDCs for 24 h.After coculture with iDCs for 24 h, the Jurkat T cells were coincubated for 15 h to analyze the IFNγ + T cells by flow cytometry (n = 3).One-way ANOVA t test, *p < 0.05 and **p < 0.01.
tumor-infiltrating CD8 + lymphocytes within the TME in locally advanced COAD and associated with poor DFS.These findings indicate that TLR1 signaling plays a critical role in the therapeutic efficacy of chemotherapy by modulating antitumor immunity.
Our previous studies reported that the extent of HMGB1 release and immune cell infiltration were associated with favorable survival outcomes in locally advanced rectal cancer patients after preoperative chemoradiotherapy treatment 26 , suggesting that the HMGB1-TLR2/4 signaling pathway may participate in chemotherapy-induced antitumor immunity.TLR2 can form heterodimers with TLR1 or TLR6 to recognize different TLR ligands, resulting in different functions 46 .Previous studies showed that activation of TLR1/2 signaling could activate the immune response, but activation of TLR2/6 signaling could suppress T-cell immunity 47,48 .Moreover, recent studies have shown that TLR1/2 agonists enhance the antitumor efficacy of CTLA-4 and PD-L1 blockade by increasing intratumoral Treg depletion 12,49 and stimulating cytotoxic T lymphocytes against tumor cells 11 .Here, our studies showed that TLR1 is essential for TLR1/2 signaling activation in DCs in response to immunogenic chemotherapy.We assumed that defective TLR1 may attenuate TLR1/2 signaling but enhance TLR2/6 signaling to suppress T-cell immunity.Therefore, HMGB1 released by immunogenic chemotherapy may activate TLR2/6 signaling in DCs to suppress T-cell-mediated immunity rather than promote the T-cell-mediated immune response in COAD patients with defective TLR1.Indeed, by silencing TLR1 expression on immature dendritic cells, we found that TLR2 signaling was less activated by HMGB1 compared to wild-type immature dendritic cells.Moreover, direct coculture of OXP-treated colorectal cancer cells with defective TLR1-iDCs showed that DC maturation and T-cell activation were remarkably attenuated in vitro, suggesting that TLR1 is essential for the HMGB1-TLR2 axis to boost antitumor immunity.Furthermore, systemic inhibition of TLR1/2 signaling markedly reduced the response to chemotherapy, leading to tumor regrowth.However, systemic administration of a TLR1/2 agonist significantly delayed tumor growth and increased the response to chemotherapy.Within the tumor microenvironment, we found that inhibition of TLR1/2 by the small molecule CU-CPT22 dramatically decreased the effect of OXP, leading to fewer tumor-infiltrating immune cells, including CD4 + , CD8 + and IFNγ + CD8 + cells.However, coadministration of a TLR1/2 agonist boosted the influence of immunogenic chemotherapy, increasing antitumor immunity to reshape the TME and eradicate residual tumors.Consistent with these observations, we found a lower density of tumor-infiltrating CD8 + lymphocytes within the tumor microenvironment in locally advanced colon carcinoma bearing the TLR1-N2418S polymorphism.Notably, we found no significant change in regulatory T (Treg) lymphocytes in these groups, which is inconsistent with previous studies 47 .Zhang  et al. found that the TLR1/2 ligand not only promoted cytotoxic T lymphocyte infiltration but also reduced the infiltration of Tregs 47 .Sharma et al. also indicated that a TLR1/2 agonist enhanced the efficacy of an anti-CTLA-4 antibody by depleting regulatory T cells and increasing IFN-γ secretion from T cells to reshape the TME in a syngeneic melanoma model 12 .We assumed that the tumor immunophenotype might be different in these models.Our syngeneic colon model CT26 had more infiltration of Tregs than the melanoma Model B16F10 50,51 , which may influence the impact of TLR1/2 agonists on the immune status within the TME.These divergences need to be investigated in different syngeneic animal models in the future.Previous reports have shown that several SNPs in TLR1 genes may modify the activation of its protein, implying that the genetic variation in the TLR1 gene might affect the innate immune response and clinical susceptibility to a wide spectrum of pathogens and confer a high risk of cancer 52 , including N248S and I602S.Here, we found that the TLR1-N248S polymorphism was not only associated with poor survival outcomes but also influenced the response to adjuvant chemotherapy.Consistent with our studies, Okazaki et al. also indicated that TLR1-I602S (rs5743618) could serve as a predictor of clinical response to FOLFIRI plus bevacizumab in patients with mCRC 15 .Moreover, Deng et al. showed that TLR1/2 expression had a positive correlation with lung cancer patient survival 46 , suggesting that TLR1/2 signaling may influence the therapeutic response to chemotherapy by modulating antitumor immunity.Therefore, low TLR1/2 expression or defective TLR1/2 signaling may result in poor clinical benefit of chemotherapy, especially immunogenic chemotherapeutic agents.
Taken together, our studies showed that the TLR1-N248S polymorphism is not only a prognostic but also a predictive factor for the clinical response to adjuvant chemotherapy in patients with locally advanced colon carcinoma.The TLR1-N248S polymorphism might reduce the ability of immunogenic chemotherapeutic agents to reshape the tumor microenvironment, leading to an insufficient antitumor immune response and tumor recurrence.

Figure 1 .
Figure 1.The association between distant metastasis-free survival (DMFS), disease-free survival (DFS) and TLR1 genotypes in locally advanced stage III COAD patients.(A) Representative images of cyto-HMGB1 expression within the tumor microenvironment in advanced colorectal cancers.(B) Kaplan-Meier curves showed that inflammation-driven cyto-HMGB1 expression was associated with poor DMFS in locally advanced COAD patients (n = 410, p = 0.0125).(C) Kaplan-Meier curves showed that inflammation-driven cyto-HMGB1 expression was associated with poor DFS in locally advanced COAD patients (n = 410, p = 0.0164).(D) The intensity of cyto-HMGB1 (H-score) within the TME in colorectal cancer patients before and after chemotherapy treatment was evaluated by IHC (n = 19, p = 0.0017, unpaired t test).(E) Kaplan-Meier curves showed that TLR1-WT (AA genotype) is associated with favorable DMFS in locally advanced COAD patients compared to TLR1-N248S (AG and GG genotypes).(F) Kaplan-Meier curves showed that TLR1-WT (AA genotype) is associated with favorable DFS in locally advanced COAD patients compared to TLR1-N248S (AG and GG genotypes).

Tumor processing to isolate tumor-infiltrating lymphocytes (TILs) and lymph nodes for flow cytometry
35The study was performed with the protocols in the Institutional Animal Care and Use Committee of China Medical University and carried out in compliance with the ARRIVE guidelines35.

Table 1 .
Clinicopathological parameters of stage II-III colon carcinoma patients (n = 869).Pearson test was used, and Fisher's exact test was used when counts were less than 5.The test did not include the "unknown or unspecified" group.
a Number of cases may differ due to missing data.* indicated p < 0.05.

Table 3 .
Univariate and multivariate analysis of DMFS and known prognostic factors in stage III colon carcinoma patients.